Reproductive Toxicology 18 (2004) 63–73
Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens) Philippe F. Kennel∗ , Catherine T. Pallen, Rémi G. Bars Bayer CropScience, Centre de Recherche de Sophia-Antipolis, BP153, F-06903 Sophia-Antipolis, France Received 23 June 2003; received in revised form 12 September 2003; accepted 2 October 2003
Abstract Three chemicals with known endocrine activities have been evaluated in the rat Hershberger assay for phase-2 of the international validation exercise within the Organization for Economic Cooperation and Development (OECD). The chemicals studied included the antiandrogens finasteride (FIN) and procymidone (PRO) and the androgen agonist 17␣-methyltestosterone (MT). Castration of sexually immature Sprague–Dawley rats was performed between post-natal days 42 and 46 whilst dosing of the chemical over 10 days was performed between post-natal days 53 and 67. Rats were co-treated with testosterone propionate (TP) for the antiandrogenic activity evaluation. The endpoints examined for evaluation of the androgenic/antiandrogenic activity were changes in sex accessory tissue (SAT) weights supplemented with measurement of testosterone and luteinizing hormone (LH) levels at sacrifice. Changes in liver, adrenal, kidney and body weights were also monitored for general toxicity assessment. Statistically significant changes in the SAT weights were detected with the three chemicals tested. Hence, the rat Hershberger assay as defined by the OECD was demonstrated sensitive enough for the detection of the endocrine disrupting activity of the three reference chemicals evaluated. © 2003 Elsevier Inc. All rights reserved. Keywords: Hershberger assay; Sex accessory tissues; Endocrine effects; Finasteride; 17␣-Methyltestosterone; Procymidone
1. Introduction In the recent years, considerable interest has been shown among both the public and the scientific community on the possibility that man-made chemicals found in the environment pose a hazard to human health and especially to reproductive function [1–7]. The endocrine-disrupting effects of many xenobiotics can be interpreted as interference with the normal regulation of reproductive processes through physiological steroid hormones. Previous data indicated that some xenobiotics bind to the androgen or estrogen receptors in target tissues [4,8]. Due to gaps in the current testing of endocrine disrupting chemicals, the US Environmental Protection Agency (US EPA) has formed the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC) which proposed a tiered screening and testing strategy to detect alterations of hypothalamic–pituitary–gonadal function, estrogen, androgen and thyroid hormone synthesis and estrogen and androgen receptor-mediated effects induced by endocrine disrupting chemicals in mammals and other taxa [9]. Many current efforts for implementing elements of this strategy are being coordinated internationally ∗ Corresponding author. Tel.: +33-492-943-447; fax: +33-493-958-454. E-mail address:
[email protected] (P.F. Kennel).
0890-6238/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2003.10.012
within the OECD. One component of the OECD in vivo screening battery is the rodent Hershberger assay. The Hershberger assay is a test method for detecting chemicals having androgenic or antiandrogenic properties, and which is characterized by the administration of test chemicals to castrated male rats for a subacute period. An assay for detecting androgenic activity in the castrated male rat has existed in various forms for almost 70 years [10–12]. The original assay was intended for the detection of androgens and was later modified for the monitoring of myotrophic properties of chemicals [13,14]. After publication of work with an extensive number of steroids evaluated for their ability to induce growth in the levator ani muscle, seminal vesicles and prostate of castrated weanling rats by Hershberger et al. in 1953 [15], the assay has subsequently been referred to as the Hershberger assay. This assay was later used in several versions to analyze both androgenic and antiandrogenic activity in rodents [16,17]. Since that time and until being considered in detail by EDSTAC and OECD, the assay has mainly been employed as a test system for antiandrogens in the rat [18–23]. The rodent Hershberger assay rely on the principle that sex accessory tissue (SAT) in the animal are under the control of androgens which stimulate and maintain their growth. If the endogenous source of these hormones is not available, either because of immaturity of
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the animal, or because the animal has been castrated, the animal requires an exogenous source to initiate and/or restore growth of these SAT. Chemicals that act as androgen agonists may be identified in the Hershberger assay if they cause an increase in the weight of the androgen-dependent SAT, or as antagonists if they cause a relative decrease when co-administered with a potent androgen [24]. There are many variations on the protocol of the assay (see [24] for review). The age and strain of the rat at castration is one major variable [24–26]. The age has ranged from the use of weanling rats through young adults to 125-day old rats [24]. The recovery period between the castration and the start of treatment has also varied, ranging from no recovery [15,18] to up to 11 days [27]. The choice, route of administration, and dosage of the reference androgen have also differed. Similarly, antiandrogens have been administered by a variety of routes and for various time periods [24]. Treatment periods have varied from 3 days [28] to 20 days [17], depending on the route of administration. Finally, the organs to be weighed and the method of weighing has also differed between the different protocols of the assay performed [24,29]. Hence, an international standardized protocol for the Hershberger assay has not yet been developed, but this assay is currently undergoing a multiphase validation process under the auspices of the OECD [30,31]. The overall aim of this validation work is to develop a robust, reliable, and relevant short-term screening assay for androgenic or antiandrogenic hormonal activity that can be considered as the basis for an OECD Test Guideline. Key issues of the validation work include definition of standardized experimental parameters of the Hershberger assay (e.g., species, age of animal, recovery period after castration, administration period, administration route of test chemicals and reference androgen, dose of reference androgen for antiandrogenicity, SAT to be weighed, method of weighing and optional endpoints such as serum hormone levels), as well as reliability and reproducibility across laboratories. The OECD Validation Management Group decided to perform the validation work in phases, taking into consideration the long use of the assay and its many variants. The phase-1 of the validation procedure has recently been finalized [32]. The interlaboratory reproducibility of measuring the weight of five androgen-responsive SAT (ventral prostate; seminal vesicles plus coagulating glands; levator ani and bulbocavernosus muscle (LABC); Cowper’s glands; glans penis) was evaluated, along with the dose-response curve for the reference androgen agonist TP and a more limited dose-response for the androgen antagonist flutamide in the presence of a fixed concentration of TP. The assay was shown to be robust and reproducible across laboratories for the detection of the potent androgen agonist (TP) and antagonist (flutamide) in the presence of several modest experimental variations. Data generated during the first phase of validation established the experimental parameters and the standard reference dose of TP to be used with androgen antagonists in phase-2.
The objective of the current phase-2 of the OECD validation exercise is to assess the protocol intra-laboratory variability and inter-laboratory reproducibility with additional chemicals, including potent and weak androgen agonists and antagonists, and to determine the relative effectiveness of the different SAT for measuring the effects [32]. At least 15 laboratories, from Europe, the US, Japan and Korea have agreed to participate in phase-2. The protocol defined for phase-2 uses peripubertal male rats castrated by removing both testes and epididymes (e.g., between 42 and 46 days of age in Sprague–Dawley rats). At the peripubertal stage of sexual development, the SAT are sensitive to androgens, having both androgen receptors and appropriate steroidogenic enzymes. The advantage of using rats at this age is that the SAT have a high sensitivity and small relative weight, which minimizes variation in responses between individual animals. The test chemical and the reference substance, where necessary, are administered in graduated doses to several groups of six male rats for 10 consecutive days and then necropsied on the 11th day approximately 24 h after the last dose. The regular data and endpoints are (i) body weight randomization in control and treatment group, (ii) daily body weight record until necropsy, (iii) daily record of mortality and clinical signs and (iv) at necropsy, weight record of the designed wet SAT (ventral prostate; seminal vesicles plus coagulating glands; LABC; Cowper’s glands; glans penis). The main optional endpoints include recording of wet liver, adrenal and kidney weights and of fixed ventral prostate weights after 24 h, and measurement of testosterone and LH circulating levels. The test chemicals selected for phase-2 of the OECD validation exercise are the androgen agonists MT and trenbolone, and the antiandrogens finasteride (FIN), procymidone (PRO), vinclozolin, linuron and p,p -DDE. The reference androgen agonist used for antiandrogenic activity evaluation is TP. Our laboratory has participated in the OECD validation exercise for the phase-2 evaluation of the Hershberger assay using FIN, PRO and MT. The results from this investigation are reported in the present study.
2. Materials and methods 2.1. General The study was performed in compliance with good laboratory practice [33] and in accordance to the French laws for animal experimentation. The protocol, in life phase, raw data and study report were subjected to specific Quality Assurance inspections. 2.2. Chemicals and dosage formulations The three chemicals (finasteride, CAS-no. 98319-26-7, purity 99.65%; procymidone, CAS-no. 32809-16-8, purity
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73
99.9%; 17␣-methyltestosterone, CAS-no. 58-18-4, purity 99.8%) and the reference androgen agonist (testosterone propionate, CAS-no. 57-85-2, purity 97%) were distributed by the chemical repository laboratory (TNO Nutrition and Food Research, He Zeist, The Netherlands) identified by the OECD for this phase-2 validation exercise. The test substances were stored in air-tight, light-resistant containers at room temperature. Dosing formulations were prepared by suspending the test chemical in an aqueous solution of methylcellulose 400 at 0.5% to produce the required dosing concentrations (w/v). The appropriate amount of reference androgen agonist (TP) was suspended (w/w) in corn oil. Formulations were prepared daily and used on the day of preparation. 2.3. Animals, housing, diet and water Male Sprague–Dawley Ico: OFA-SD (IOPS Caw) rats were obtained from Charles River Laboratories (L’Arbresle, France). Animals were castrated by the supplier at peripuberty between post-natal days 42 and 46 (the postnatal age being calculated with the day of birth being defined as postnatal day zero). The rats were acclimatized to laboratory conditions for five to six days prior to treatment and were 53–57 days old at the start of exposure to the test chemical. Animals were assigned permanent identification numbers within groups using a randomization procedure that ensured a similar body weight distribution among groups. In each study, body weights were within ± 20% of the mean body weight on the day of randomization. Animals were in a weight range from 194 to 260 g on the first day of treatment. They were housed individually in suspended stainless steel wire mesh cages. No bedding material was used. The laboratory conditions in the study room were controlled and monitored by an automatic system. The target specifications were a temperature of 22 ± 2 ◦ C, a relative humidity of approximately 55%, a 12 h light/dark regime and an air exchange rate of 15 per hour. Certified rodent pelleted and irradiated diet (reference A04C-10, Scientific Animal Food and Engineering, Villemoisson-sur-Orge, France) and filtered and softened tap water from the municipal water supply were available ad libitum. 2.4. Experimental design The experimental design followed the OECD phase-2 protocol [32] recommended by the lead laboratory of this validation exercise (US EPA National Health and Environmental Effect Research Laboratory, Endocrinology Branch, Research Triangle Park, NC, US). Three separate experiments with six castrated rats per group were conducted. The antiandrogenic activity of FIN at 25, 5, 1 and 0.2 mg/kg per day and of PRO at 100, 30, 10 and 3 mg/kg per day was evaluated in Experiments 1 and 2, respectively. The androgenic activity of MT at 40, 10, 2 and 0.5 mg/kg per day
65
was evaluated in Experiment 3. The dose levels of the three chemicals and that of TP (0.4 mg/kg per day) were recommended by the OECD Validation Management Group [32]. In Experiments 1 and 2, animals were co-administered the test chemical by gavage and the TP by subcutaneous (SC) injection on the dorsal surface on a daily basis for 10 days. A reference control group with androgenic stimulation consisted of castrated rats given TP by the SC route and an aqueous solution of methylcellulose 400 at 0.5% by gavage daily for 10 days. In addition, a control check for the androgenic activity of TP was run, whereby a vehicle control group of animals received corn oil by the SC route and an aqueous solution of methylcellulose 400 at 0.5% by gavage daily for 10 days. In Experiment 3, animals were given MT by gavage on a daily basis for 10 days. A reference control group consisted of castrated rats given an aqueous solution of methylcellulose 400 at 0.5% by gavage daily for 10 days. All animals were dosed at 5 ml/kg body weight per day for treatment administered by the oral route and at 0.5 ml/kg body weight per day for TP administered by the SC route. The oral route by gavage for the chemicals and the SC route for TP were recommended by the OECD Validation Management Group [32]. Treatment by the oral and SC routes were administered consecutively. The dosage was adjusted daily for body weight change. 2.5. General observations Clinical signs were recorded at least once daily for all animals. The nature, onset, severity, reversibility and duration of clinical signs were recorded. Preputial separation was examined before the first day of treatment and on the day of necropsy. 2.6. Body weight and food consumption Individual body weight was recorded daily throughout the treatment period and prior to necropsy. Food consumption was measured at weekly intervals. 2.7. Hormone analysis Blood samples were collected from the abdominal aorta immediately prior to necropsy. LH and testosterone hormone levels were determined on individual plasma samples with specific radio-immunoassay kits (Amersham, Les Ulis, France, for LH and Beckman Coulter, Villepinte, France, for testosterone). 2.8. Post mortem examinations and organ weights All animals were euthanatized by exsanguination under pentobarbital anesthesia approximately 24 h after the last dose administration. Animals were not fasted prior to
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sacrifice. The order in which animals were necropsied was randomized in each experiment. A first random permutation was performed on groups and then a second random permutation was performed on animals such that one animal from each group was processed before necropsy of the next animal from each group. The liver, kidneys and adrenal glands were collected and weighed. The SAT (ventral prostate, seminal vesicles with coagulating glands, LABC, glans penis and Cowper’s glands) were carefully dissected free from adhering fat and then weighed (paired organs were weighed together). The ventral prostate was fixed for 24 h in 10% neutral-buffered formalin and then weighed again wet. 2.9. Statistical analysis OECD phase-2 protocol recommended that variations in body weight should be both experimentally and statistically controlled. At study initiation, animals were assigned to the groups using a randomization procedure which ensured homogeneity of body weights between groups. However, treatment with androgenic or antiandrogenic chemicals could affect body weight. To detect any direct treatment effect on organ and SAT weights over and above any indirect effects caused by the effect of the treatment on body weight, both absolute means and means adjusted by the covariance analysis (ANCOVA) were analyzed [34]. Group variances were intercompared by the use of Bartlett test for homogeneity of variances. If Bartlett test indicated homogeneous variances, the exposed group means were compared to the control mean using the Dunnett test (two-sided). If Bartlett test indicated heterogeneous variances, data (body weight gains excepted) were transformed using a log or a square root transformation to stabilize the variances. If the Bartlett test on transformed data was not significant, the Dunnett test was performed on transformed data. If the Bartlett test indicated heterogeneous variances (even after data transformations), the non-parametric Mann–Whitney test (two-sided) was performed. Comparisons of variances between the control and the TP treated groups were performed using the F test of homogeneity of variances. When the F test was not significant, means were compared using the t-test (two-sided). When the F test was significant, data (body weight gains excepted) were transformed using a log or a square root transformation and the t-test was applied on transformed data. If the F test remained significant even after transformation, means were compared using the Mann-Whitney test (two-sided). In order to take into account variations in organ and SAT weights in relationship to body weight changes, the covariance analysis was performed on these parameters using the terminal body weight as covariable and adjusted means were compared using the Dunnett test. Means were compared at the 5 and 1% levels of signification. All the statistical procedures were conducted using SAS programs (SAS Software Release 8.2, SAS Institute Inc., Cary, NC, USA).
3. Results 3.1. General observations No mortality was observed in any of the three experiments. Food consumption in all treated groups was comparable to the control values (data not shown). In Experiment 1, no treatment-related clinical signs were observed. In Experiment 2, treatment-related clinical signs consisted of white area on eyes noted on days 3–5 until necropsy in approximately half of the animals co-treated with PRO plus TP, irrespective of the dose level of the androgen antagonist. In Experiment 3, treatment-related clinical signs consisted of white area on eyes, starting on day 2 or 3 and continuing until day 11 in 4/6 animals, and of lacrimation or ocular discharge observed between days 1 and 11 in one animal at 40 mg/kg per day of MT. White area on eyes was also noted, starting on days 5–8 and continuing until day 11, in 4/6 animals at 10 mg/kg per day of MT. 3.2. Body weights In Experiments 1 and 2, overall body weight gain was statistically significantly increased in the reference control groups treated with TP (+44 and +26%, respectively), when compared to the corresponding vehicle control groups (data not shown). FIN at 25, 5, 1 or 0.2 mg/kg per day plus TP had no effect on TP-induced increase in overall body weight gain, whilst PRO at 100, 30, 10 or 3 mg/kg per day plus TP induced a slight dose-related decrease in overall body weight gain (−12, −12, −7 and −3%, respectively). In Experiment 3, MT at 40, 10, 2 or 0.5 mg/kg per day induced no changes in the overall body weight gain when compared to the reference vehicle control group. In all three experiments, terminal body weight of the chemical-treated groups was similar to those of the respective reference control group at each of the dose levels tested (Tables 1 and 2). 3.3. Organ weights In Experiments 1 and 2, absolute liver weight was statistically significantly increased in the reference control groups treated with TP in both experiments (+15 and +13%, respectively), absolute kidney weight was statistically significantly increased in the reference control groups treated with TP in Experiment 1 (+18%) and absolute adrenal gland weight was slightly reduced in Experiment 2, when compared to the corresponding vehicle control group (Table 1). However, the increase in absolute liver and kidney weights observed was most likely related to the overall increase in body weight induced by the reference androgen agonist TP, as the covariance analysis showed that adjusted liver and kidney weights in the reference control groups treated with TP were similar to the values of the corresponding vehicle control group (data not shown). FIN at 25, 5, 1 or 0.2 mg/kg
Exp
Body weights (g) ± S.D.
Absolute organ weights (g) ± S.D.
Absolute sex accessory tissue weights (mg) ± S.D.
Gavage Test agent (mg/kg per day)
SC route Androgen (mg/kg per day)
Initial
Liver
Seminal vesicle
1 2 3 4 5 6
Vehicle Vehicle FIN (0.2) FIN (1) FIN (5) FIN (25)
Vehicle TP (0.4) TP (0.4) TP (0.4) TP (0.4) TP (0.4)
211.1 211.3 212.0 208.8 209.5 211.7
± ± ± ± ± ±
4.8 9.0 10.5 4.2 6.8 6.3
273.3 301.3 287.8 288.5 284.0 293.2
± ± ± ± ± ±
5.5 20.7# 18.7 12.6 21.4 6.9
10.6 12.2 11.5 11.6 11.3 12.5
± ± ± ± ± ±
0.8 1.0# 1.6 1.5 1.0 0.7
1.86 2.19 1.95 1.99 1.96 2.01
± ± ± ± ± ±
0.14 0.15## 0.19 0.14 0.21 0.11
0.063 0.055 0.055 0.046 0.056 0.052
± ± ± ± ± ±
0.011 0.008 0.010 0.012 0.004 0.011
25 316 186 89 102 63
± ± ± ± ± ±
8 108## 50∗∗ 58∗∗ 39∗∗ 51∗∗
82 301 286 255 255 248
± ± ± ± ± ±
16 36## 21 12 19 47∗
48 80 73 72 70 70
± ± ± ± ± ±
4 6## 7 9 9 16
2 29 24 16 15 10
± ± ± ± ± ±
1 9## 3 10∗∗ 5∗∗ 5∗∗
13 133 116 67 54 43
± ± ± ± ± ±
4 36## 22 27∗∗ 16∗∗ 13∗∗
20 181 153 94 74 62
± ± ± ± ± ±
2 55## 31 38∗∗ 16∗∗ 11∗∗
1 2 3 4 5 6
Vehicle Vehicle PRO (3) PRO (10) PRO (30) PRO (100)
Vehicle TP (0.4) TP (0.4) TP (0.4) TP (0.4) TP (0.4)
219.4 218.1 218.9 214.7 222.8 216.6
± ± ± ± ± ±
12.0 13.6 7.5 11.9 9.9 14.3
290.2 306.8 305.2 296.8 301.2 294.8
± ± ± ± ± ±
16.3 17.6 7.1 16.4 16.4 17.0
11.9 13.4 13.1 12.5 13.8 13.9
± ± ± ± ± ±
0.8 0.8## 0.8 1.0 1.6 0.8
2.08 2.23 2.17 2.14 2.17 2.08
± ± ± ± ± ±
0.20 0.20 0.13 0.10 0.11 0.10
0.068 0.054 0.063 0.062 0.066 0.081
± ± ± ± ± ±
0.017 0.009 0.014 0.010 0.006 0.014∗∗
37 356 362 406 216 146
± ± ± ± ± ±
13 53## 47 176 59∗∗ 70∗∗
74 293 340 322 278 204
± ± ± ± ± ±
20 42## 34 79 40 32∗
43 79 83 86 80 67
± ± ± ± ± ±
9 5## 20 8 11 19
4 33 30 31 29 16
± ± ± ± ± ±
1 8## 8 9 14 5∗
16 174 180 151 103 53
± ± ± ± ± ±
9 33## 43 52 17∗∗ 24∗∗
23 257 260 218 147 82
± ± ± ± ± ±
10 44## 69 66 28∗∗ 30∗∗
Age at castration (PND)
Group
1
42–44
2
46
Treatment
Terminal
Significantly different from vehicle control group (group 1) at P < 0.05. Significantly different from vehicle control group (group 1) at P < 0.01. ∗ Significantly different from vehicle + TP control group (group 2) at P < 0.05. ∗∗ Significantly different from vehicle + TP control group (group 2) at P < 0.01. #
##
Kidney
Adrenal gland
LABC
Glans penis
Cowper’s glands
Ventral prostrate (fresh)
Ventral prostrate (fixed)
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73
Table 1 Body weights and sex accessory tissue weights from Sprague–Dawley castrated rats given 10 consecutive daily treatments with the indicated antiandrogen (n = 6 animals/group)
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68
Experiment
3
∗
Age at castration (PND)
Group
46
1 2 3 4 5
Treatment
Body weights (g) ± S.D.
Absolute organ weights (g) ± S.D.
Absolute sex accessory tissue weights (mg) ± S.D.
Gavage Test agent (mg/kg per day)
Initial
Liver
Seminal vesicle
Vehicle MT (0.5) MT (2) MT (10) MT (40)
240.9 242.4 239.5 239.0 243.6
Terminal
± ± ± ± ±
12.5 9.9 13.0 16.0 9.4
301.0 306.8 296.8 303.7 309.3
Significantly different from vehicle control group (group 1) at P < 0.05. Significantly different from vehicle control group (group 1) at P < 0.01. a n = 5. ∗∗
± ± ± ± ±
22.6 14.9 13.4 21.4 11.6
11.6 11.9 11.7 11.8 13.1
Kidney
± ± ± ± ±
0.9 1.1 1.0 0.7 1.0
2.06 2.13 2.07 2.13 2.19
± ± ± ± ±
Adrenal gland
0.21 0.18 0.18 0.08 0.18
0.066 0.061 0.051 0.062 0.054
± ± ± ± ±
0.012 0.010 0.008 * 0.008 0.007
53 55 45 78 142
± ± ± ± ±
29 16 17 39 27∗∗
LABC
124 113 119 159 263
± ± ± ± ±
Glans penis 27 14 22 17∗ 23∗∗
52 55 45 68 79
± ± ± ± ±
Cowper’s glands 7 7 13 16 11∗∗
6 4 6 10 19
± ± ± ± ±
2 1 2 5 4∗∗
Ventral prostrate (fresh) 16 17 22 50 110
± ± ± ± ±
6 8 11 25∗∗ 35∗∗
Ventral prostrate (fixed) 23 20 27 74 142
± ± ± ± ±
19 8 18 34∗∗ 49∗∗,a
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73
Table 2 Body weights and absolute sex accessory tissue weights from Sprague–Dawley castrated rats given 10 consecutive daily treatments with the indicated androgen agonist (n = 6 animals per group)
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73
per day plus TP and PRO at 100, 30, 10 or 3 mg/kg per day plus TP had no significant effect on TP-induced changes in absolute liver, kidney and adrenal gland weights, with the exception of a statistically significant increase in absolute adrenal gland weight (+50%) noted at the high dose of PRO plus TP (Table 1). The covariance analysis corroborated the result (data not shown), thus indicating a slight systemic toxicity at the high dose of PRO plus TP. In Experiment 3, MT at 40 mg/kg per day induced a slight not statistically significant increase in absolute liver weight (+13%) and a slight not statistically significant decrease in mean adrenal gland weight (−18%), when compared to the reference vehicle control groups (Table 2). The covariance analysis also showed changes in adjusted organ weights (data not shown), thus indicating a slight systemic toxicity in this high dose group. MT at 10, 2 or 0.5 mg/kg per day induced no changes in absolute and adjusted liver, kidney and adrenal gland weights, with the exception of a statistically significant decrease in absolute and adjusted adrenal gland weight in the 2 mg/kg per day dose group, but this change was considered not to be treatment-related (Table 2). Hence, overall a slight systemic toxicity was noted at the high dose of PRO (i.e., 100 mg/kg per day) and of MT (i.e., 40 mg/kg per day). 3.4. Sex accessory tissue weights In all three experiments, examination of the glans penis showed that Sprague–Dawley rats castrated between post-natal days 42 and 46 had not systematically achieved their preputial separation. As the dissection of glans penis may be affected in case of incomplete preputial separation, comparison of glans penis weight with data derived from these animals may not be accurate and should be considered with caution. In Experiments 1 and 2, a highly statistically significant increase in all absolute SAT weights was detected in the reference control groups treated with TP (Experiment 1: +1164% for seminal vesicles, +267% for LABC, +67% for glans penis, +1350% for Cowper’s glands, +923% for ventral prostate and +805% for ventral prostate fixed; Experiment 2: +862% for seminal vesicles, +296% for LABC, +84% for glans penis, +725% for Cowper’s glands, +988% for ventral prostate and +1017% for ventral prostate fixed), when compared to the corresponding vehicle control group (Table 1). Results of the covariance analysis led to the same conclusion (data not shown). Thus, the increase in all absolute SAT weights was clearly attributable to the reference androgen TP. FIN at 25, 5, 1 or 0.2 mg/kg per day plus TP attenuated the TP-induced increase in SAT weights, as a dose-related decrease in all absolute SAT weights was observed (Table 1). The difference was statistically significant for absolute seminal vesicle, LABC, Cowper’s gland and ventral prostate (fresh or fixed) weights at 25 mg/kg per day, for absolute seminal vesicle, Cowper’s gland and ventral prostate (fresh or fixed) weights at 5 and 1 mg/kg
69
per day, and only for absolute seminal vesicle weights at 0.2 mg/kg per day, when compared to the values of the reference control group treated with TP. The covariance analysis confirmed all the statistical significances detected (data not shown). Thus, the significant decreases in absolute SAT weights were clearly related to the specific antiandrogenic effect of FIN. PRO at 100 mg/kg per day plus TP attenuated the TP-induced increase in SAT weights, as a decrease in all absolute SAT weights was observed (Table 1). The difference was statistically significant for absolute seminal vesicle, LABC, Cowper’s gland and ventral prostate (fresh or fixed) weights, when compared to the values of the reference control group treated with TP. The covariance analysis confirmed all the statistical significances detected (data not shown). Thus, regardless of the slight systemic toxicity noted at this high dose level, the significant decreases in absolute SAT weights were clearly related to the specific antiandrogenic effect of PRO. PRO at 30 mg/kg per day plus TP also attenuated the TP-induced increase in SAT weights, as a statistically significant decrease in absolute seminal vesicle and ventral prostate (fresh or fixed) weights was observed (Table 1). On the whole, the covariance analysis confirmed the statistical significances detected (data not shown). Hence, the significant decreases in absolute seminal vesicle and ventral prostate weights were also attributable to the specific antiandrogenic effect of PRO at 30 mg/kg per day. PRO at 10 mg/kg per day plus TP induced a slight non-statistically significant attenuation of the TP-induced increase in absolute and adjusted ventral prostate (fresh or fixed) weight (Table 1). Other mean absolute and adjusted SAT weights were similar to the reference control group treated with TP. PRO at 3 mg/kg per day plus TP had no effect on TP-induced increase in absolute or adjusted SAT weights (Table 1). In Experiment 3, MT at 40 mg/kg per day induced a statistically significant increase in all absolute SAT weights, when compared to the reference vehicle control groups (Table 2). The covariance analysis confirmed this result (data not shown). Thus, regardless of the slight systemic toxicity noted at this high dose level, the significant increases in absolute SAT weights were attributable to the specific androgenic effect of MT. MT at 10 mg/kg per day also induced an increase in all absolute SAT weights (Table 2), but the difference was statistically significant only for absolute LABC and ventral prostate (fresh or fixed) weights, when compared to the reference vehicle control group. The covariance analysis confirmed the statistical significances detected (data not shown). Thus, the significant increases in absolute LABC and ventral prostate weights were also attributable to the specific androgenic effect of MT at 10 mg/kg per day. MT at 2 mg/kg per day induced a slight non statistically significant increase in absolute and adjusted ventral prostate (fresh or fixed) weight, when compared to the reference vehicle control groups (Table 2). Other mean absolute and adjusted SAT weights were similar to the reference vehicle control group. MT at 0.5 mg/kg per day induced no changes in absolute and adjusted SAT
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Table 3 Testosterone and LH plasma levels from Sprague–Dawley castrated rats given 10 consecutive daily treatments with the indicated antiandrogen (n = 6 animals/group) Experiment
1
Age at castration (PND)
42–44
2
46
Group
Hormone levels (ng/ml) ± S.D.
Treatment Gavage Test agent (mg/kg per day)
SC route Androgen (mg/kg per day)
Testosterone
LH
1 2 3 4 5 6
Vehicle Vehicle FIN (0.2) FIN (1) FIN (5) FIN (25)
Vehicle TP (0.4) TP (0.4) TP (0.4) TP (0.4) TP (0.4)
0.31 1.06 1.17 1.05 1.13 0.86
± ± ± ± ± ±
0.10a 0.28##,a 0.30 0.26 0.30 0.42
9.12 2.02 5.02 4.58 2.25 8.10
± ± ± ± ± ±
4.68 1.30#,b 3.14a 4.23a 1.24 9.99
1 2 3 4 5 6
Vehicle Vehicle PRO (3) PRO (10) PRO (30) PRO (100)
Vehicle TP (0.4) TP (0.4) TP (0.4) TP (0.4) TP (0.4)
0.28 0.96 1.12 1.16 1.25 1.25
± ± ± ± ± ±
0.12a 0.31## 0.39 0.57 0.30 0.36
9.02 0.89 2.40 4.74 4.56 8.93
± ± ± ± ± ±
3.41 0.47##,a 2.37 3.32∗∗,a 2.43∗∗ 5.45∗∗
Significantly different from vehicle control group (group 1) at P < 0.05. Significantly different from vehicle control group (group 1) at P < 0.01. ∗∗ Significantly different from vehicle + TP control group (group 2) at P < 0.01. a n = 5. b n = 4. #
##
weights, when compared to the reference vehicle control groups (Table 2). In the three experiments performed in this study, fixation of the ventral prostate for tissue weight comparison has no impact on the result. Indeed, even if the tissue weights were heavier, the statistical analysis of the fixed ventral prostate yielded exactly the same statistical significances as the fresh ventral prostate weights and led to the same conclusions.
or 0.2 mg/kg per day plus TP had no or little effect on TP-induced changes in testosterone and LH plasma levels (Table 3). A non-statistically significant increase in LH plasma level was noted in the high dose group of FIN plus TP, but this increase was mainly due to a particularly high value observed in one single animal. PRO at 100, 30, 10 and 3 mg/kg per day plus TP induced a dose-related increase in mean LH plasma level (+903, +412, +433 and +170%, respectively), the difference being statistically significant for LH plasma level at 100, 30 and 10 mg/kg per day of PRO plus TP, when compared to the reference control animals treated with TP alone (Table 3). No change was observed in testosterone level in animals treated with PRO plus TP. In Experiment 3, at 40 mg/kg per day of MT, an apparent increase in mean testosterone plasma level (+37%) and an apparent decrease in mean LH plasma level (−38%) was noted, although not statistically significant, when compared to the values of the control animals (Table 4). MT at 10,
3.5. Hormone levels In Experiments 1 and 2 (Table 3), TP administration induced a statistically significant increase in testosterone plasma level (+242 and +243%, respectively) and a subsequent statistically significant decrease in LH plasma level (−78 and −90%, respectively), when compared to the corresponding vehicle control group. FIN at 25, 5, 1
Table 4 Testosterone and LH plasma levels from Sprague–Dawley castrated rats given 10 consecutive daily treatments with the indicated androgen agonist (n = 6 animals/group) Experiment
3
∗ Significantly
n = 5. b n = 3. c n = 4. a
Age at castration (PND)
Group
46
1 2 3 4 5
Treatment
Hormone levels (ng/ml) ± S.D.
Gavage Test agent (mg/kg per day)
Testosterone
LH
Vehicle MT (0.5) MT (2) MT (10) MT (40)
0.30 0.34 0.20 0.26 0.41
± ± ± ± ±
8.61 12.8 7.99 7.96 5.33
different from vehicle control group (group 1) at P < 0.05.
∗∗ Significantly
0.08a 0.11b 0.04c 0.15a 0.16
± ± ± ± ±
2.40 7.67 3.08a 3.30 2.61c
different from vehicle control group (group 1) at P < 0.01.
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73
2 or 0.5 mg/kg per day induced no or little changes in testosterone and LH plasma levels.
4. Discussion A current issue for regulatory agencies at the international level is endocrine-related modes of action including those mediated by the estrogen or androgen receptor. One initiative by the EDSTAC was the recommendation of a tiered series of in vitro and in vivo protocols for the assessment of such modes of action [9]. In mammalian species, these protocols include the castrated male rat assay described by Hershberger et al. [15]. The rodent Hershberger assay has been used extensively over the years and many modifications to the basic protocol have been described (see Section 1). The advantage of this assay, compared to other in vivo procedures, is that it is fairly simple, short-term, and relatively specific for direct screening of androgenic or antiandrogenic effects [35]. Although this assay has been in wide use since the middle of the 20th century, it has not been implemented in toxicology testing as a standardized screening tool for endocrine active chemicals. For this purpose, the OECD Validation Management Group has undertaken an international multiphase validation exercise of the Hershberger assay. The reliability and feasibility of this assay is presently being investigated on a worldwide scale by at least 15 laboratories using 7 reference chemicals known to interact with the endocrine system through different mechanisms. Our laboratory has participated in the current phase-2 of the OECD validation exercise to evaluate the ability of the Hershberger assay to detect the antiandrogenic activity of FIN and PRO and the androgenic activity of MT, using the experimental conditions identified by the OECD (see Section 1). The results from this investigation are reported herein with a view to contributing to the definition of a standard protocol by the OECD for routine use in toxicology testing. For the evaluation of the antiandrogenic activity of FIN and PRO, SAT weights of castrated male rats co-treated with the antiandrogen plus TP for 10 days, were compared to those of a reference control group treated with TP alone. As expected for both antiandrogen evaluations, the control group for androgenic activity produced a consistent statistically significant increase in the weight of the five androgen-responsive SAT (ventral prostate, seminal vesicles, LABC, Cowper’s glands and glans penis). FIN, a 5␣-reductase inhibitor, showed a potent antiandrogenic activity. The growth of the SAT stimulated by TP was attenuated in a dose-related manner by FIN and this was statistically significant in seminal vesicles, LABC, Cowper’s gland and ventral prostate at 25 mg/kg per day, in seminal vesicles, Cowper’s glands and ventral prostate at 5 and 1 mg/kg per day, and in seminal vesicles at 0.2 mg/kg per day. The antiandrogenic activity of FIN was most apparent in terms of a decrease in seminal vesicle, Cowper’s gland and ventral prostate weights. This observation corroborates the fact
71
that these tissues rely on 5␣-reductase to convert testosterone to the more active molecule 5␣-dihydrotestosterone, thus being more sensitive to the inhibitory action of FIN. It has been reported that the growth of the LABC muscles is testosterone dependent, rather than 5␣-dihydrotestosterone dependent, and that 5␣-reductase inhibitors should less affect these muscles [36]. In our study, FIN attenuated statistically significantly the regrowth of the LABC muscles only at the high dose level, in agreement with previous findings obtained in similar conditions [24]. PRO, a dicarboximide fungicide, also showed a potent antiandrogenic activity in our study. The growth of the SAT stimulated by TP was attenuated in a dose-related manner by PRO and this was statistically significant in seminal vesicle, LABC, Cowper’s gland and ventral prostate at 100 mg/kg per day and in seminal vesicles and ventral prostate at 30 mg/kg per day. This result corroborates previous findings where dicarboximide fungicides were shown to be active in a similar Hershberger assay [37]. The evaluation of androgenic activity of MT demonstrated a clear dose-related trophic response in the SAT. The weight was statistically significantly increased in the five androgen-responsive SAT at 40 mg/kg per day and in LABC and ventral prostate at 10 mg/kg per day. Previous Hershberger assays to detect androgenic activity of MT showed increase in some SAT at doses as low as 0.5 mg/kg per day [38], 25 mg/kg per day [24], or 100 mg/kg per day [39] with slightly differing experimental conditions. In all the three experiments of the present study, glans penis weight may not have been very accurate as taken from tissue with variable preputial separation status. Indeed, examination of glans penis showed that Sprague–Dawley rats castrated between post-natal days 42 and 46 had not systematically achieved preputial separation, thus making difficult the glans penis dissection. In the present study, this limitation has no impact on the interpretation of the result, but this issue should be considered by the OECD Validation Management Group at the time of data compilation and compared to the results generated with identical and different rat strains before defining a standard Hershberger protocol for routine use in toxicology testing. The present Hershberger assay was capable of detecting a clear dose-related androgen antagonist effect on SAT weights with FIN and PRO, and a clear dose-related androgen agonist effect with MT, taking into account only the mandatory endpoints of the OECD phase-2 protocol. The optional endpoints provided supplemental information further confirming the chemical-mediated endocrine effects and could help to reveal its mechanism of action. In the present study, fixation of the ventral prostate for tissue weight comparison had no impact on the ability to detect the effects of the three chemicals evaluated. Indeed, even if the tissue weights were heavier, the statistical analysis of the fixed ventral prostate provided exactly the same kind of results as the fresh ventral prostate weights. These findings indicated that formalin fixation of the ventral prostate did not interfere with interpretation of assay results. The optional liver,
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kidney and adrenal gland weights were assessed with clinical signs, body weight and food consumption as indices of systemic toxicity. In general, FIN, PRO or MT induced no treatment-related changes in liver, kidney and adrenal gland weights, with the exception of a marked increase in adrenal gland weight at the high dose of PRO. Such effect of PRO or other androgen receptor antagonists on adrenal gland weight and function has already been described [40]. Hence, in the three experiments, the doses tested had no severe adverse effects on liver, kidney or adrenal gland weights, or on clinical signs or body weight change. Therefore, the doses of FIN, PRO and MT selected by the OECD Validation Management Group were appropriate for determining whether these chemicals interfere specifically with androgen-mediated mechanisms in vivo. The determination of testosterone and LH plasma levels were only optional endpoints, because the method of collection of blood samples is not standardized between laboratories and a number of critical factors are frequently not adequately controlled in the study design, factors such as the circadian fluctuations, the effect of acute stress, and other environmental influences. In our study, blood sampling was performed within a 4 h window interval between 8:45 a.m. and 12:45 p.m. As expected for both antiandrogen evaluations, the control for androgenic activity (TP by the SC route) induced a consistent statistically significant increase in mean testosterone plasma level and a subsequent statistically significant decrease in mean LH plasma level. FIN had no or little effect on TP-induced changes in testosterone and LH plasma levels, whilst PRO induced a dose-related increase in LH plasma levels, the difference being statistically significant at 100, 30 and 10 mg/kg per day of PRO. The increase in circulating LH levels is likely due to a blockage of the androgen receptors by PRO at the hypothalamic-pituitary level, which prevents the normal feedback mechanism that should have taken place with the administered TP to control the circulating level of LH [41]. In the present study, the inhibition of TP feedback was almost complete at the high dose of PRO, as the LH plasma level was increased up to the level observed in control castrated animals. In a previous report, Nellemann et al. [37] observed statistically significant increase in LH plasma only at dose levels of 25 mg/kg per day and above, in a similar Hershberger assay. The higher sensitivity observed in our study may be due to the longer duration of the treatment (i.e., 10 days versus 7 days). In the experiment using MT, only a tendency towards an increase in testosterone plasma level and a decrease in LH plasma level was noted in the high dose group, but this additional observation confirmed the androgen-receptor agonism activity of MT clearly detected on SAT weights. Overall, with the exception of the statistically significant LH change detected with PRO, the treatments failed to alter significantly hormone levels within 24 h after the last dose even though SAT weights were affected. These hormone data confirm previous findings showing that, in contrast to the organ weight measurements, sexually immature rats are less sensitive than sexually ma-
ture rats for detecting compound-induced hormonal changes [42–44]. In conclusion, the Hershberger assay, as defined by the OECD for the phase-2 validation exercise, was sensitive enough to detect a dose-related change in SAT weights of castrated rats treated with the 5␣-reductase inhibitor FIN, the weak antiandrogen PRO or the potent androgen agonist MT. The optional endpoints contained in the phase-2 protocol provided additional information on the toxicity induced by the test chemical and helped to provide a better understanding on how the test chemical mediates its effect. The findings of the present study, taken together with those of other participating laboratories of this OECD phase-2 validation exercise, will contribute to the definition of a standard Hershberger protocol for routine use in toxicology testing.
Acknowledgments The authors would like to thank Mrs. Valerie Cipolla for her excellent technical assistance and Dr. Sheila Wason for critical review of this manuscript. The authors would also like to express their gratitude to the animal care and necropsy staffs. This investigation was made possible by funds from the Endocrine Modulator Study Group.
References [1] Colborn T, Vom Saal FS, Sato AM. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 1993;101:378–84. [2] Crisp TM, Clegg ED, Cooper RL, et al. Environmental endocrine disruption: an effects assessment and analysis. Environ Health Perspect 1998;106(Suppl 1):11–56. [3] Gray Jr LE. Xenoendocrine disrupters: laboratory studies on male reproductive effects. Toxicol Lett 1998;102/103:331–5. [4] Kelce WR, Wilson FM. Environmental antiandrogens: developmental effects, molecular mechanisms, and clinical implications. J Mol Med 1997;75:198–207. [5] McLachlan JA. Functional toxicology: a new approach to detect functionally active xenobiotics. Environ Health Perspect 1993;101: 582–7. [6] McLachlan JA, Korach KS. Estrogens in the environmental global health implications. Environ Health Perspect 1995;103:3–4. [7] Sharpe RM. Hormones and testis development and the possible adverse effects of environmental chemicals. Toxicol Lett 2001;120:221–32. [8] Danzo BJ. The effects of environmental hormones on reproduction. Cell Mol Life Sci 1998;54:1249–64. [9] EPA. Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). Final report EPA/743/R-98/003; 1998. [10] Callow RK, Deanesly R. Effect of androsterone and of male hormone concentrate on the accessory reproductive organs of castrated rats, mice and guinea-pigs. Biochem J 1935;29:1424–45. [11] Korenchevsky V. The assay of testicular hormone preparations. Biochem J 1932;26:413–22. [12] Korenchevsky V, Dennison M, Schalit R. The response of castrated male rats to the injection of the testicular hormone. Biochem J 1932;26:1306–14.
P.F. Kennel et al. / Reproductive Toxicology 18 (2004) 63–73 [13] Eisenberg E, Gordan GS, Elliott HM. Testosterone and tissue respiration of the castrate male rat with a possible test for myotrophic activity. Endocrincology 1949;45:113–9. [14] Eisenberg E, Gordan GS. The levator ani muscle of the rat as an index of myotrophic activity of steroidal hormones. J Pharmacol Exp Therap 1950;99:38–44. [15] Hershberger LG, Shipley EG, Meyer RK. Myotrophic activity of 19-nortestosterone and other steroids determined by modified levator ani muscle methods. Proc Soc Exp Biol Med 1953;83:175–80. [16] Dorfman RI. Androgens and anabolic agents. In: Dorfman RI, editor. Methods in hormone research. vol. IIA. New York: Academic Press; 1969. p. 151–220. [17] Dorfman RI. Antiandrogens. In: Dorfman RI, editor. Methods in hormone research. vol. IIA. New York: Academic Press; 1969. p. 221–49. [18] Kelce WR, Lambright CR, Gray LE, Roberts KP. Vinclozolin and p,p -DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen receptor-mediated mechanism. Toxicol Appl Pharmacol 1997;142:192–200. [19] Raynaud JP, Bouton MM, Moguilewsky M, et al. Steroid hormone receptors and pharmacology. J Steroid Biochem 1980;12:143–57. [20] Rittmaster RS, Magor KE, Manning AP, Norman RW, Lazier CB. Differential effect of 5␣-reductase inhibition and castration on androgen-regulated gene expression in rat prostate. Mol Endocrinol 1991;5:1023–9. [21] Shao TC, Kong A, Cunningham GH. Effects of 4-MAPC, a 5␣-reductase inhibitor, and cyproterone acetate on regrowth of the rat ventral prostate. Prostate 1994;24:212–20. [22] Snyder BW, Winneker RC, Batzold FH. Endocrine profile of WIN 49596 in the rat: a novel androgen receptor antagonist. J Steroid Biochem 1989;33:1127–32. [23] Wakeling AE, Furr BJA, Glen AT, Hughes LR. Receptor binding and biological activity of steroidal and nonsteroidal antiandrogens. J Steroid Biochem 1981;15:355–9. [24] Ashby J, Lefevre PA. Preliminary evaluation of the major protocol variables for the Hershberger castrated male rat assay for the detection of androgens, antiandrogens, and metabolic modulators. Reg Toxicol Pharmacol 2000;31:92–105. [25] Yamada T, Kunimatsu T, Sako H, et al. Comparative evaluation of a 5-day Hershberger assay utilizing mature male rats and a pubertal male assay for detection of flutamide’s antiandrogenic activity. Toxicol Sci 2000;53:289–96. [26] Yamasaki K, Sawaki M, Takatsuki M. Strain sensitivity differences in the Hershberger assay. Reprod Toxicol 2001;15:437–40. [27] O’Connor JC, Frame SR, Davis LG, Cook JC. Detection of the environmental antiandrogen p,p -DDE in CD and Long-Evans rats using a Tier I screening battery and a Hershberger assay. Toxicol Sci 1999;51:44–53. [28] Peets EA, Henson MF, Neri H. On the mechanism of the antiandrogenic action of flutamide (␣-␣-␣-trifiuoro-2-methyl-4 -nitrom-propionotoluidide) in the rat. Endocrinology 1973;94:407–532. [29] Yamada T, Sunami O, Kunimatsu T, et al. Dissection and weighing of accessory sex glands after formalin fixation, and a 5-day assay using young mature rats are reliable and feasible in the Hershberger assay. Toxicology 2001;162:103–19. [30] OECD, Organization for Economic Cooperation and Development. 2nd meeting of the Validation Management Group on screening
[31]
[32]
[33]
[34] [35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
73
and testing for endocrine disrupters (mammalian effects). Paris; 2000. OECD, Organization for Economic Cooperation and Development. Proceedings of the third meeting of the Validation Management Group for the screening and testing of endocrine disrupters (mammalian effects). Joint meeting of the chemicals committee and the working party on chemicals, pesticides and biotechnology. Paris; 2001. OECD, Organization for Economic Cooperation and Development. Final OECD report of the work towards the validation of the rat Hershberger assay: phase−1, androgenic response to testosterone propionate, and anti−androgenic effects of flutamide. ENV/JM/TG/ EDTA(2002)1/REV2. Paris; 2002. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, No. 1. Paris: Organization for Economic Cooperation and Development; 1998. Eryl S. The analysis of organ weight data. Toxicology 1977;8:13–22. Gray Jr LE, Kelce WR, Wiese T, et al. Endocrine screening methods workshop report: detection of estrogenic and androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mechanisms. Reprod Toxicol 1997;11:719–50. Blohm TR, Laughlin ME, Benson HD, et al. Pharmacological induction of 5␣-reductase deficiency in the rat: separation of testosterone-mediated and 5␣-dihydrotestosterone-mediated effects. Endocrinology 1986;119:959–66. Nellemann C, Dalgaard M, Lam HR, Vinggaard AM. The combined effects of vinclozolin and procymidone do not deviate from expected additivity in vitro and in vivo. Toxicol Sci 2003;71:251–62. Yamasaki K, Takeyoshi M, Sawaki M, Imatanaka N, Shinoda K, Takatsuki M. Immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals. Toxicology 2003;183:93–115. Sunami O, Kunimatsu T, Yamada T, et al. Evaluation of a 5-day Hershberger assay using young mature male rats: methyltestosterone and p,p -DDE, but not fenitrothion, exhibited androgenic or antiandrogenic activity in vivo. J Toxicol Sci 2000;25:403–15. Andrews P, Freyberger A, Hartmann E, et al. Feasibility and potential gains of enhancing the subacute rat study protocol (OECD test guideline no. 407) by additional parameters selected to determine endocrine modulation. A pre-validation study to determine endocrine-mediated effects of the antiandrogenic drug flutamide. Arch Toxicol 2001;75:65–73. Hosokawa S, Murakami M, Ineyama M, et al. The affinity of procymidone to androgen receptor in rats and mice. J Toxicol Sci 1993;18:83–93. Cook JC, Mulline LS, Frame SR, Biegel LB. Investigation of a mechanism for Leydig cell tumorigenesis by linuron in rats. Toxicol Appl Pharmacol 1993;119:195–204. Viguier-Martinez MC, Hochereau de Reviers MT, Barenton B, Perreau C. Effect of a non-steroidal antiandrogen, flutamide, on the hypothalamo-pituitary axis, genital tract and testis in immature rats: endocrinological and histological data. Acta Endocrinol 1983;102:299–306. Viguier-Martinez MC, Hochereau de Reviers MT, Barenton B, Perreau C. Endocrinological and histological changes induced by flutamide treatment on the hypothalamo-hypophyseal testicular axis of the adult male rat and their incidences on fertility. Acta Endocrinol 1983;104:246–52.